(CANCER RESEARCH 49, 4204-4209. August I. 1989) Relationship of Reduced Folate Changes to Inhibition of DNA Synthesis Induced by Methotrexate in LI 210 Cells in Vivo1

D. G. Priest,2 Marlene Bunni, and F. M. Sirotnak

Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425 [D. G. P., M. B.], and Memorial Sloan- Kettering Cancer Center, New York, New York 10021 [F. M. S.J

ABSTRACT be adequate and more elaborate mechanisms involving other reduced folate pools and direct effects on folate-dependent Reduced folate levels and DNA synthesis were examined in LI 210 biosynthetic enzymes may be necessary to fully explain the cells in mice after exposure to a wide range of methotrexate doses. A radioenzymatic assay based upon entrapment of tissue 5,10-methylene- cytotoxicity of MTX. However, it has also been shown recently tetrahydrofolate (( II-I-II4), and other reduced folates after cycling to that reduced folates in the LI210 system grown in vitro depend this form, into a stable ternary complex with thymidylate synthase and strongly upon media folate levels with regard to endogenous |3H|-5-fluoro-2'-deoxyuridine 5'-monophosphate was used to estimate levels of total reduced folates, the distribution among reduced reduced folates. DNA synthesis was estimated from incorporation of folates, and the response of folates to MTX (2). Hence, an in [5H)-2'-deoxyuridine into DNA. The predominant reduced folate in cells vivo model system was considered for similar studies that pro from untreated animals was 10-formyltetrahydrofolate (IO-O1OKII.,; vides a more natural environment for tumor growth with regard 3.59 pniol/KI" cells). The four other reduced folates measured, tetra- to folate type and level and is thus more therapeutically relevant. hydrofolate (III,), < 11 I IL, dihydrofolate (III.), and 5-methyltetrahy- To overcome analytical problems associated with evaluation of drofolate (5-< 11,!•114),were present in nearly equal amounts yielding a total reduced folate level of 6.24 pmnl/IO" cells. When methotrexate was reduced folate levels in cells grown in this manner, a ternary complex assay method, based on entrapment of CH2FH4 into administered s.c. at doses of 1.5, 12, and 400 mg/kg, the level of Hl: a stable complex with thymidylate synthase and ['HJFdUMP increased dramatically and total tetrahydrofolates measured decreased extensively within 1 h. DNA synthesis was completely inhibited during (11, 12), was used. Results are discussed in terms of potential the first 1-2 h after administration of each dose of methotrexate with biochemical models for the cytotoxic effects of MTX on tumor onset of recovery after 4 and 20 h at 1.5 and 12 mg/kg and not at all cells. after the highest dose. Both PH* and O U 11, were extensively depleted at 12 and 400 mg/kg methotrexate but considerably less depletion of MATERIALS AND METHODS ( II..MI4, and none of MI4, was observed at 1.5 mg/kg during a period when DNA synthesis was essentially abolished. The metabolically linked [3H]FdUMP (20 Ci/mmol) was purchased from Moravek Biochem- 5-( 11,1114and IO-( 11()l 11, pools were also extensively depleted follow icals, Brea, CA, and [-"HldUrd (20 Ci/mmol) was purchased from New ing treatment with methotrexate. While Ml. levels expanded extensively England Nuclear, Boston, MA. MTX was purchased from Lederle after drug treatment, the total increase did not account for the extent of Laboratories, Pearl River, NY, and FH4 was obtained from Fluka depletion of the combined tetrahydrofolate pools. The change in concen Chemical Co., Ronkonkoma, NY. 10-CHOFH4 was a gift from Dr. tration with time of any one folate pool was apparently not sufficient to John McGuire, Roswell Park, Buffalo, NY. FH2, 5-CH3FH4, NADPH, explain completely the duration of inhibition of DNA synthesis; however, and other reagents were purchased from Sigma Chemical Company, sustained inhibition of DNA synthesis was generally associated with St. Louis, MO. Thymidylate synthase (0.9 unit/mg) and dihydrofolate maintenance of an expanded Ml. pool and delay in repletion of the reductase (24 units/mg) were purified from aminopterin-resistant Liu combined tetrahydrofolate pools. Discussion is presented with respect to tobacillus casei cells according to methods described previously (13, the impact of these results on changing notions of the mode of action of 14). Pig liver methylenetetrahydrofolate reductase (0.25 unit/mg) was classical antifolates. purified according to the procedure of Matthews and Haywood (15) and 10-formyltetrahydrofolate deacylase (0.01 unit/mg) was prepared by the method of Huennekens and Scrimgeor (16). INTRODUCTION Transplantation of the L1210 cells into female C57BL x DBA/2 F, Because MTX' is a potent inhibitor of dihydrofolate reduc (hereafter called 151)21,) mice was conducted as previously described (17). Typically, IO4cells were injected into the intraperitoneal cavity. íase(1) and gives rise to inhibition of intracellular thymidylate When cell density reached 200 x IO6 cells/animal, MTX was admin (2) and DNA (3) synthesis, the impact of this drug on cellular istered s.c. at three different doses, 1.5, 12, and 400 mg/kg. Tumor reduced folates has been the focus for interpretation of its cells were harvested from the peritoneal cavity of animals and washed growth-inhibitory properties. Classically, it has been proposed once with cold 0.175 M NH4C1 and once with cold buffered 0.14 M that the dihydrofolate reductase product FH4 and the closely NaCl-0.01 M sodium phosphate, pH 7.4. Cells were centrifugally pel related thymidylate synthase substrate, CH:FH4, are suffi leted from this buffer and stored at —¿70°Cuntilanalyzed. Cell concen ciently depleted in the presence of MTX to cause disruption of tration was determined densitometrically (18). Protein concentration thymidylate synthesis (4-7). However, direct measurement of was estimated by the method of Bradford (19) and used to confirm cell concentration. reduced folates in cell systems grown in culture (2, 8-10) have Measurement of ['I I|

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1989 American Association for Cancer Research. METHOTREXATE EFFECTS ON FOLATES IN VIVO detected with assays currently available. These methods are based upon Table 1 Reduced folate concentration and efficiency of standard folate recovery the entrapment of CH:>FH4by thymidylate synthase and ['H]FdUMP from LI210 cells grown in viro Reduced folates were determined by the ternary complex assay from cells to form a stable ternary complex (22). Cells were suspended in an harvested 5 days after mice were inoculated i.p. with IO4 cells each. Values extraction buffer that contained 50 IHM Tris-HCl, 50 mM sodium represent the mean ±SEM from five separate experiments in which cells from ascorbate, 1 mM EDTA, and 0.25 M sucrose, pH 7.4. The final concen 2-4 animals were combined. Recovery of standard reduced folates was conducted tration of cells ranged from 20 to 30 x IO*1cells/ml. Cells were boiled by introduction of each standard into cell suspensions prepared from animals that had received a 400-mg/kg MTX dose for 4 h. The level of each was for 3 min to achieve lysis and prevent enzymatic cycling during assay. Extracts were centrifuged at 10,000 x g for 10 min at 4°C.CH2FH4 approximately equal to that found endogenously. Each sample was then subjected to the entire lysis and assay process. Recovery was calculated after subtraction of was estimated by combining aliquots (20-60 //I) of cell-free extract with endogenous reduced folate in each case. Results represent the mean of 6 deter 10 milliunits of thymidylate synthase and 125 n\i ['HJFdUMP (20 Ci/ minations •¿SEM. mmol) in a total volume of 200 p\ of the above extraction buffer. Under folate recovery these conditions tissue extract < 11 I 11, is sufficiently limiting to assure (pmol/10'cells)0.48 incorporation of the entire pool even though longer chain length (%)98.7 polyglutamylated forms bind more avidly (23). Ternary complex for CH,FH4 ±0.13 ±7.1 mation was allowed to proceed at 25°Cfor 30 min, stopped by the FH4 0.80 ±0.11 88.9 ±1.3 FH2 0.66 ±0.24 70.4 ±5.5 addition of 1% sodium dodecyl sulfate, and boiled for 5 min. Aliquots 5-CH,FH4 0.71 ±0.23 77.5 ±8.5 (25 ¿il)werecentrifugally filtered over minicolumns of Sephadex G-25 10-CHOFH4Concentration3.59 ±1.02Standard 84.7 ±6.4 to separate bound from free ['HJFdUMP and counted on a Packard scintillation counter to determine bound ['HJFdUMP (12). For meas urement of the four other reduced folates a separate aliquot (20-60 n\) from the same cell extract was used. FH4 was determined by the same 100 - reaction system except in the presence of 6.5 mM formaldehyde (11). 5-CHjFH4 was determined following enzymatic conversion to CH:FH4 in the presence of 1.1 milliunits methylenetetrahydrofolate reducíase (24). 10-CHOFHj was estimated after enzymatic conversion to CH2FH4 in the presence of 1.0 milliunit 10-formyltetrahydrofolate deacylase, 6.5 mM formaldehyde, and 0.15 mM NADPH (25). FH2 was determined after enzymatic cycling to CH;FH4 in the presence of 35 milliunits dihydrofolate reducíase,6.5 mM formaldehyde, and 0.15 mM NADPH (26). The specificity of FH; measurements were verified by the introduction of 50 nM MTX which resulted in a complete block of FH2 recovery.

RESULTS 20 The ternary complex entrapment approach was used to esti Time (hr) Fig. 1. Effect of MTX on ['HJdUrd incorporation into DNA of 1.1210 cells mate five reduced folate pools in LI210 cells grown in BD2F, grown in viro. ('Hjdllrd (2 /¿Ci)was administered i.p. into animals which had mice. It can be seen in Table 1 that replicate analysis of five received MTX doses of 1.5 (CD).'2 (•),and 400 (A) mg/kg. After 10 min cells separate samples prepared by combining cells harvested from were harvested and incorporation of radioactivity into DNA determined. Points, two to four control animals, yielded estimates of less than 1 average of 3 determinations, expressed as percentage of control values. pmol/106 cells for all pools except 10-CHOFH4. Total reduced folate, based on the sum of these five pools, was 6.24 pmol/106 Fig. 1 that incorporation is rapidly abolished within 2 h at each cells. The average error for all pools in these control experi of the three doses; however, the time required for recovery of ments was 28% which was similar to the overall average error DNA synthesis is highly dose dependent. In the case of the calculated from all measurements. Variability for three separate lowest dose (1.5 mg/kg) inhibition is still essentially complete animals which were maintained under identical conditions at 4 h but recovery to near 90% of the pretreatment level has yielded an average range of values for all pools of 31%. occurred by 7 h. At the intermediate dose (12 mg/kg) inhibition A further source of error considered was accumulation of remains complete until approximately 20 h. At the highest dose dUMP during MTX treatment (27) or interference from the (400 mg/kg) recovery does not occur. The lack of recovery of antifolate itself. Hence, recovery experiments were conducted DNA synthesis at 400 mg/kg and the onset of recovery at 20 h in which reference reduced folates were added to cells harvested at the 12-mg/kg dose have been reported previously (3) but from animals that had received the highest MTX dose. It can additional data at these doses were included here for purposes be seen in Table 1 that recovery was very close to quantitative of comparison and to confirm earlier results obtained with this for the primary folate of interest (CH2FH4), was approximately experimental system. 85% for the major folate (10-CHOFH4), but ranged to as low The mechanism often proposed by which MTX inhibits DNA as 70% for the FH2 pool. Recovery results were not significantly synthesis and cell growth is depletion of FH4, and in turn the different when conducted with control cells or when standards cofactor for thymidylate synthase CH:FH4, as a result of dihy were added subsequent to lysis and extracts reboiled to mimic drofolate reducíaseinhibition (4-7). It can be seen in Fig. 2 the lysis procedure. Because recovery of individual folate pools thai ihe iwo highesl levels of MTX led lo early and nearly is greater than 70% in the worst case and very near quantitative complete depletion of both CH2FH4 (Fig. 2A) and FH4 (Fig. in the case of CH2FH4, no normalization of estimates has been 2B). These two pools returned to levels which were 60-70% of applied to results. conlrol values by 4 h and generally remained Ihere Ihroughoul To examine the impact of MTX on the five reduced folate Ihe experiment except somewhat elevated levels may occur at pools in Table 1, the drug was introduced s.c. at three widely 24 h for Ihe 12-mg/kg MTX dose. Al the lowest MTX level different doses, 1.5, 12, and 400 mg/kg. These doses were Ihere is much less depletion of CH2FH4 during the 1-4 h perior! (Fig. 2A) and no significant change in FH4 (Fig. IB) '~u.nig intended to yield minimal, intermediate, and profound effects, respectively, on DNA synthesis (3, 28, 29). It can be seen in this period, inhibition of DNA synthesis is essentially complete 4205

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1989 American Association for Cancer Research. METHOTREXATE EFFECTS ON FOLATES M VÃŒVO

largest pool in untreated cells, was rapidly and profoundly depleted at all MTX doses (Fig. 35) and only at the lowest dose, did repletion occur (16 h after drug administration). Nevertheless, neither changes in this pool nor those in the 5- CH3FH4 pool showed a consistent correlation with the duration of inhibition of DNA synthesis. Dihydrofolate reducíase inhibition by MTX would be ex pected to result in accumulation of FH2 (5). It can be seen in Fig. 4 that substantial accumulation occurred at all three levels of MTX used. In all cases there is a rapid rise and subsequent fall of this reduced folate which generally mimics intracellular MTX levels (3). Only at the lowest MTX dose (Fig. 4A) does the level return to pretreatment values. However, there is no 10 20 apparent correlation between the time at which this pretreat Time (hr) ment level is reached and the resumption of DNA synthesis Fig. 2. Effect of MTX on CH¡FH4(A) and FH4 (B) in L1210 cells grown in vivo. MTX was introduced s.c. into animals with a tumor burden of 200 x 10' (see Fig. 1). A correlation is similarly lacking between FH2 cells/animal at doses of 1.5 (D), 12 (•),and 400 (A) mg/kg. Cells from 2-4 levels and the duration of inhibition of DNA synthesis following animals were harvested at each time point and the ternary complex assay was the two higher MTX doses (Fig. 4, B and C). used to determine 111, and lililí, in the presence and absence of formaldehyde, respectively. Points, average of 4-6 determinations; ears, SEM. Since changes in the individual pools of reduced folates were not consistent with the duration of inhibition of DNA synthesis, consideration was given to changes in combined tetrahydrofo- late pools (FH4, CH2FH4, 5-CH,FH4, 10-CHOFH4). These results are also shown in Fig. 4 along with the grand total which includes FH2 for comparison. Although depletion of the four tetrahydrofolates occurred at all doses of MTX, depletion was generally greater following 12 mg/kg and greatest following 400 mg/kg. Also, substantial repletion of these four pools can be seen at 1.5 mg/kg, while less occurred at 12 mg/kg MTX and very little at 400 mg/kg MTX. Total reduced folate levels also fell in a somewhat dose-dependent manner and remained relatively constant throughout the experiment. It is unclear whether this depletion results from accumulation of a reduced folate undetectable by the methods used or reflects a true general loss of intracellular folate polyglutamate in response to the drug.

6.0 Fig. 3. Effect of MTX on 5-CHjFH4(/() and 10-CHOFH, (B) in LI 210 cells o 4.0 grown in vivo. MTX was introduced s.c. into animals at doses of 1.5 (D), 12 (•), u and 400 (A) mg/kg. 5-CH3FH4 and 10-CHOFH4 were determined by the ternary complex assay in the presence of methylenetetrahydrofolate reducíaseand 10- formyltetrahydrofolate deacylase, respectively. Other conditions are the same as o those described in Fig. 2. Points, average of 4-6 determinations; bars, SEM. n . 0- a. (see Fig. 1). Thus, while a diminished level of the thymidylate o 6.0 ra synthase substrate CH I-H., could play an important role in the k 4MJ5 4.0 interruption of DNA synthesis, total depletion of this pool is e o not essential. Further, the time at which this pool recovers to c o pretreatment levels does not correspond precisely to resumption O of DNA synthesis. In view of this behavior of CH2FH4 and the S £ lack of the predicted depletion of the FH., pool, the possible o 6.0 influence of other pools was investigated. •¿o e 4.0 Since both the FH4 and CH2FH4 pools are integrally linked o -O3 metabolically to other reduced folate pools, it was reasonable 2.0 O) to anticipate that these other pools can have an influence on OC the intracellular level of FH4 and CH2FH4 and their response to MTX. Two such pools that are directly linked to the FH4 10 20 and CH2FH4 pools are 5-CH,FH4 and 10-CHOFH4. Further Time (hr) more, these two pools have been shown previously to respond Fig. 4. Effect of MTX on FH; and other reduced folates in LI 210 cells grown in vivo. MTX was introduced s.c. at doses of 1.5 (A ), 12 (B), and 400 (C) mg/kg. to MTX in the L1210 system in vitro (2). Thus, they were FHz (D) was determined by the ternary complex assay in the presence of dih\ dro evaluated and the results are shown in Fig. 3. In both cases, tolate reducíase.The combined tetrahydrofolate pool (•)is the sum of \ \\,. depletion was nearly immediate and very extensive. In the case CH¡FH4,5-CHjFH4, and 10-CHOFH,, determined as described in Figs. 2 and 3. of 5-CHjFH4 (Fig. 3,4), a general upward trend can be seen by Total reduced folate (O) is the sum of the combined tetrahydrofolate pools and III. Conditions are the same as those described in Fig. 2. Points, average of 4 16-24 h that is similar for all MTX doses. 10-CHOFH4, the 6 determinations; bars, SEM. 4206

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1989 American Association for Cancer Research. METHOTREXATE EFFECTS ON FOLATES IN VIVO

In summary, these results show that in general the onset of ever, monoglutamate forms of these other reduced folates were inhibition of DNA synthesis in LI210 cells is associated with used because polyglutamylated forms were unavailable and extensive depletion of the combined tetra hyd rotolai e pools and hence some undetectable interference could nevertheless occur rapid expansion in the level of HI. Although the duration of from cycling of the natural polyglutamylated forms of other inhibition of DNA synthesis at different doses of MTX shows reduced folates. A further potential problem associated with some correlation with the level and time of repletion of the this methodology which was considered is interference from combined tetrahydrofolate pools, neither these gross changes dUMP which could accumulate in the presence of high MTX nor changes in the CH2FH4 pool alone can by themselves fully doses (27). Interference could arise from this source because explain the duration of DNA synthesis inhibition; thus addi introduction of thymidylate synthase and FdUMP into extracts tional factors could also play a role. causes a competition to be established between dUMP and FdUMP for available thymidylate synthase sites. In the pres ence of tissue CH2FH4, successful FdUMP binding leads to a DISCUSSION stable ternary complex. However, when dUMP binds, enzyme In general, intracellular reduced folates, because they exist at turnover can occur which results in depletion of the target pool several different oxidation states with regard to both the pteri- and its concomitant underestimation. Hence, any system in dine ring and the one-carbon unit (30) and because they contain which dUMP accumulation is suspected should be tested for pol yglutam ates with up to seven residues (31), provide a large recovery of added reference CH2FH4 to detect assay interference array of closely related compounds that must be regulated to from the presence of the nucleotide. In the present case there maintain metabolic integrity and cell survival in the presence is either little dUMP accumulation or the level of FdUMP used of cytotoxic antifolates. Study of individual folate metabolizing in the assay and the timing of addition of assay components enzymes has contributed to a better understanding of this successfully overcome potential interference from its presence, regulation, but the sheer number of enzymes involved, each because very near quantitative recovery of reference CH2FH4 with its own unique set of regulatory properties, taken in was obtained (see Table 1). conjunction with the number of reduced folate species with Reduced folate pools in I.I210 cells grown in vitro have which they interact, make evaluation of the regulation of indi shown a strong dependence on media folate level with regard vidual intracellular folate cofactor concentrations from isolated to both total reduced folate concentration and distribution enzyme studies very difficult. An alternative approach is direct among the individual pools (2). Interestingly, total reduced folate levels in L1210 cells grown in vivo (6.24 pmol/106 cells) estimation of intracellular reduced folates. The primary diffi culty with this approach is analytical problems associated with were slightly higher than in cells cultured in the classical RPMI 1640 media (3.5 pmol/106 cells) which contains 2.3 ¿¿Mmedia the number and instability of reduced folates typically present in the highly complex intracellular environment (30). Chro folate (2). The distribution among the five folates examined in matographie separation techniques have been applied success vivo also showed some similarities to the in vitro system in that fully to several systems (8, 32-35). Unfortunately, cell extracts the FH4, CH2FH4, and FH2 pools are relatively low and the 10- must be pretreated to chemically or enzymatically remove CHOFH4 pool was relatively high. The primary difference polyglutamates to reduce the number of species present and between the in vivo and in vitro LI210 system is the 5-CH3FH4 this has made detection of more unstable folates difficult. pool. The in vitro system showed a relatively high level of this Furthermore, the Chromatographie approach typically requires folate which was similar to the level of 10-CHOFH4, whereas prior uniform radiolabeling of folate pools to obtain sufficient in the i/i vivo system the 5-CH,FH4 pool was much lower and detection sensitivity, and thus in vivo experiments are extremely the 10-CHOFH4 pool dominated (57%). The underlying reason difficult since isotope dilution and/or loss throughout the entire for these differences must be in part related to both the level animal can occur. and type of extracellular folate to which cells are exposed in The ternary complex assay, based upon entrapment of the two different systems. However, other environmental con CH2FH4 by thymidylate synthase and [3H]FdUMP, provides ditions would also be expected to play a role. the enzymatic selectivity to directly isolate a single folate species MTX has been shown to lead to diminished thymidylate (2) without prior removal of polyglutamates and without prior and DNA (3) synthesis and probably exerts its cytotoxic effects folate pool radiolabeling since the second ligand, | ÕI|Idl MP, in this manner. In this in vivo L1210 system, MTX accumulates provides the radioactive sensitivity (12). Potential problems rapidly in a dose-dependent manner and within a very brief period gives rise to nearly complete cessation of ['H]dUrd that can be associated with the ternary complex assay system primarily center around unwanted cycling reactions that can incorporation into DNA (3). In general, accumulation of FH2 occur as a result of enzyme activities in cell lysates. Because and depletion of other reduced folate pools (except in the case entrapment of CH2FH4 into the ternary complex is highly of FH4 at the lowest drug dose) also occur rapidly, the former efficient (22), there is a tendency for other reduced folates to tending to mirror MTX accumulation (3). After the initial cycle into this pool when necessary enzymes and cofactors are accumulation phase, intracellular MTX decreases rapidly. The present during the complexation procedure. To overcome this depletion time course depends upon the total MTX dose and difficulty a brief (3-min) boiling step was introduced that de level achieved. DNA synthesis resumes when intracellular MTX natures the potentially interfering enzymes and prevents this approaches the level of dihydrofolate reductase present in the cycling but also can potentially lead to chemical instability of cell. Since the time at which DNA synthesis resumes after each some intracellular folates (36). This instability is the most likely of the three different MTX doses used in this study varies reason for the less than quantitative recovery of some folates widely, these data provide an opportunity to evaluate the rela (see Table 1). Further, overestimation of a particular folate, tionship between reduced folate levels and the effect of this which could result from the incorporation of an extraneous antifolate on this proliferative function. pool into a target pool during cycling reactions, was shown to Attempts to attribute MTX effects on DNA synthesis to be negligible by intentional contamination with reference folates changes in individual reduced folate pools were not entirely during previous in vitro studies of the L1210 system (2). How satisfactory. While depletion of FH4 and CH2FH4 was extensive 4207

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1989 American Association for Cancer Research. METHOTREXATE EFFECTS ON FOLATES IN VIVO at higher MTX doses, there was less depletion of CH2FH4 and provide tumor growth conditions which more closely mimic the none of FH4 at the lowest MTX level even though DNA clinical situation and hence are of greater pharmacological synthesis was abolished for at least two hours. Hence, total relevance. depletion of the thymidylate synthase substrate CH2FH4 was not essential to explain DNA synthesis interruption. The same ACKNOWLEDGMENTS general conclusion was reached in the in vitro LI210 system (2). Nevertheless, the fact that a relatively strong depletion of The authors express their appreciation to Barbara Rembiesa and these pools occurred, at least at high MTX levels, as had been Lauri Mines for their technical assistance. classically predicted (4-7), suggests that the change in level of these two pools plays an important role in MTX cytotoxicity although changes in other folate pools could also contribute. It REFERENCES is not possible to evaluate the impact of changes in the other 1. Werkheiser, W. C. The biochemical, cellular, and pharmacological action three pools, FH2, 10-CHOFH4, and 5-CH,FH4, during the and effects of the folie acid antagonists. Cancer Res., 23: 1277-1285,1963. onset of inhibition because effects were already maximal at the 2. Bunni, M., Doig, M. T., Donato, H., Kesavan, V., and Priest, D. G. Role of first data point obtained. methylenetetrahydrofolate depletion in methotrexate-mediated intracellular thymidylate synthesis inhibition in cultured LI210 cells. Cancer Res., 48: Although the FH2 pool expanded in response to MTX in 3398-3404, 1988. every case, the time dependence for the maintenance of this 3. Sirotnak, F. M., Donsbach, R. C, Dorick, D. M., and Moccio, D. M. Tissue pharmacokinetics, inhibition of DNA synthesis, and tumor cell kill after expansion did not always correspond directly to the effects on high-dose methotrexate in murine tumor models. Cancer Res., 36: 4672- DNA synthesis. A more comprehensive approach to the role of 4678, 1976. FH2 accumulation in DNA synthesis inhibition is that it be 4. Jackson, R. C. Biological effects of folie acid antagonists with antineoplastic activity. Pharmacol. Ther., 25:61-82, 1984. viewed concomitant with other reduced folate pools that can 5. Jackson, R. C., and Grindey, G. B. The biochemical basis for methotrexate compete for the same enzymic sites as this folate. That is, FH2 cytotoxicity. In: F. M. Sirotnak, J. J. Burchall, W. B. Ensminger, and J. A. Montgomery (eds.). Folate Antagonists as Therapeutic Agents, Vol. 1, pp. not only competes with MTX for dihydrofolate reducíasesites 289-315. New York: Academic Press, 1984. but it also is known to inhibit several other reduced folate- 6. Jolivet, J., Cowan, K. H., Curt, G. A., Clendeninn, N. J., and Chabner, B. metabolizing enzymes including, for example, thymidylate syn A. The pharmacology and clinical use of methotrexate. N. Engl. J. Med., 309: 1094-1104, 1983. thase (37) and methylenetetrahydrofolate reducíase(38). In the 7. Moran, R. G., Mulkins, M., and Heidelberger, C. Role of thymidylate case of both of these enzymes, the substrate with which FH2 synthetase activity in development of methotrexate cytotoxicity. Proc. Nati. must compete is CH2FH4. Hence, the concentration of this Acad. Sci. USA, 76: 5924-5928, 1979. 8. Allegra, C. J., Fine, R. L. Drake, J. C., and Chabner, B. A. The effect of substrate should be considered simultaneously with FH in methotrexate on intracellular folate pools in human MCF-7 breast cancer regulatory models which explain MTX effects on reduced folate cells: evidence for direct inhibition of purine synthesis. J. Biol. ( 'hem.. 267: metabolism and cytotoxicity. This same rationale can be ex 6478-6485, 1986. 9. Allegra, C. J., Drake, J. C., Jolivet, J., and Chabner, B. A. Inhibition of tended to other folate pools and their respective enzymes. phosphoribosylaminoimidazolecarboxamide transformylase by methotrexate However, all reduced folates are metabolically linked and re and dihydrofolic acid polyglutamates. Proc. Nati. Acad. Sci. USA, 82:4881- 4885, 1985. spond to MTX in concert as can be seen by the fact that MTX 10. Matherly, L. H., Barlowe, C. K., Phillips, V. M., and Goldman, I. D. The causes a concomitant depletion of the four tetrahydrofolate effects on 4-aminoantifolates on 5-formyltetrahydrofolate metabolism in pools and accumulation of FH . Thus, apparently, as one pool L1210 cells. J. Biol. Chem., 262:710-717, 1987. 11. Priest, D. G., Veronee, C. D., Mangum, M., Bednarek, J. M., and Doig, M. is depleted, other pools attempt to restore that pool as long as T. Comparison of folylpolyglutamate hydrolases of mouse liver, kidney, linkage enzymes and adequate cofactors are available. This muscle and brain. Mol. Cell. Biochem., 43: 81-87, 1982. 12. Priest, D. G., and Doig. M. T. Tissue folate polyglutamate chain-length explains the failure to totally deplete a pool such as CH2FH4. determination by electrophoresis as thymidylate synthase-fluorodeoxyuridy- Since FH2 is isolated following the MTX block of dihydrofolate late ternary complexes. Methods Enzymol., 122: 313-319, 1986. reducíasebecause it can be depleted only by this enzyme and 13. Dunlap, R. B., Harding, N. G. L., and Huennekens, F. M. Thymidylate synthetase from amethopterin-resistant Lactobacillus casei. Biochemistry, can accumulate only through the thymidylate synthase reaction, 10: 88-97, 1971. levels of this folate theoretically may provide the most unam 14. Galivan, J. H., Maley, G. F., and Maley, F. The effect of substrate analogs biguous assessment of MTX effects based on a single pool. on the circular dichroic spectra of thymidylate synthetase from Lactobacillus casei. Biochemistry, 14: 3338-3344, 1975. However, it must be recognized that this is an imperfect corre 15. Matthews, R. G., and Haywood, B. J. Inhibition of pig liver methylene lation because, for example, very high levels of MTX probably tetrahydrofolate reducíasebydihydrofolate: some mechanistic and regulatory implications. Biochemistry, IS: 4845-4851, 1979. also block the thymidylate synthase reaction and hence prevent 16. Huennekens, F. M., and Scrimgeor, K. G. N'°-Formyltetrahydrofolic de- accumulation of FH2 to a level which might otherwise be acylase Methods Enzymol., 6: 373-375, 1963. achieved. This is suggested by the results in Fig. 4 in which the 17. Hutchinson, D. J.. Robinson, D. L., Martin, D., Ittensohn, O. L., and Dillenberg, J. Effects of selected anticancer drugs on the survival time of intermediate MTX dose gives rise to a greater accumulation of mice with 1.1210 leukemia: relative response of amimetabolite-resistam FH than the highest dose. strains. Cancer Res., 22: 57-72, 1962. A comparison of the L1210 system in vivo with that in vitro 18. Chello, P. L., Sirotnak, F. M., and Dorick, D. M. Alterations in the kinetics of methotrexate transport during growth of L12 IO murine leukemia cells in (2) with regard to the effects of MTX on reduced folate pools culture. Mol. Pharmacol., 18: 274-280, 1980. shows that in general FH accumulates and other reduced 19. Bradford. M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. folates are depleted in response to MTX in both cases but the Anal. Biochem., 72: 248-254, 1976. degree to which FH2 is dominant with respect to other pools is 20. Margolis, S., Philips, F. S., and Sternberg, S. S. The cytotoxicity of metho much greater in the in vivo system. These disparate results may trexate in mouse small intestine in relation to inhibition of folie acid reducíase and of DNA synthesis. Cancer Res., 31: 2037-2046, 1971. arise from differences in both the level and type of reduced 21. Phillips, F. S., Sirotnak, F. M., Sodergren, J. E., and Hutchison, D. J. Uptake folate in the environment in which cells are grown and the of methotrexate, aminopterin and methasquin and inhibition of dihydrofolate reductase and of DNA synthesis in mouse small intestine. Cancer Res., 33: difference in exposure of these cells to MTX. However, other 153-158, 1973. parameters may also play a role. Because of these differences 22. Santi, D. V., McHenry, C. S., and Perriard, E. R. A filter assay for thymi observed between the in vivo and in vitro systems, detailed dylate synthetase using 5-fluoro-2'-deoxyuridylate as an active site titrant. Biochemistry, 13:467-470, 1974. assessment of the mechanism of action of MTX may require 23. Priest, D. G., and Mangum, M. Relative affinity of 5,10-methylenetetrahy- relatively extensive use of in vivo experiments in the future to drofolate polyglutamates for the Lactobacillus casei thymidylate synthetase- 4208

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1989 American Association for Cancer Research. METHOTREXATE EFFECTS ON FOLATES ¡NVIVO

5-fluorodeoxyuridylate binary complex. Arch. Biochem. Biophys., 210:118- 32. Kashani, S. A., and Cooper, B. A. Endogenous folate of normal fibroblasts 123, 1981. using high-performance liquid chromatography and modified extraction pro 24. Doig, M. T., Peters, J. R., Sur, P., Dang, M., and Priest, D. G. Determination cedure. Anal. Biochem., 146: 40-47, 1985. of mouse liver S-methyltetrahydrofolate concentration and polyglutamate 33. Moran, R. G., Werkheiser, W. C., and Zakrzewski, S. F. Folate metabolism forms. J. Biochem. Biophys. Methods, 10: 287-294, 1985. in mammalian cells in culture. I. Partial characterization of the folate 25. Kesavan, V., Doig, M. T., and Priest, D. G. A radioenzymatic method for derivatives present in 1.121(1 mouse leukemia cells. J. Biol. Chem., 251: the determination of tissue 10-formyltetrahydrofolate. J. Biochem. Biophys. 3569-3575, 1976. Methods, 12: 311-317, 1986. 34. Matherly, L. H., Barlowe, C. K., and Goldman, I. D. Antifolate polyglu- 26. Doig, M. T., Sur, P., Peters, J. R., and Priest, D. G. Enzymatic determination tamylation and competitive drug displacement at dihydrofolate reducíaseas of tissue dihydrofolate. Anal. Lett., 17:2067-2073, 1984. important elements in leucovorin rescue in LI210 cells. Cancer Res., 46: 27. Goulian, M., Bleile, B., and Tseng, B. Y. The effect of methotrexate on levels 588-593, 1986. of dUTP in animal cells. J. Biol. Chem., 255:10630-10637, 1980. 35. Duch, D. S., Bowers, S. W., and Nichol, C. A. Analysis of folate cofactor 28. Sirotnak, F. M., and Donsbach, R. C. The intracellular concentration de levels in tissues using high-performance liquid chromatography. Anal. pendence of antifolate inhibition of DNA synthesis in LI 210 leukemia cells. Biochem., 130: 385-392, 1983. Cancer Res., 34:3332-3340, 1974. 36. Wilson, S. D., and Horne, D. W. Evaluation of ascorbic acid in protecting 29. Sirotnak, F. M., Donsbach, R. C., Moccio, D. M., and Dorick, D. M. labile folie acid derivatives. Proc. Nati. Acad. Sci. USA, SO: 6500-6504, Biochemical and pharmacokinetic effects of leucovorin after high-dose meth 1983. otrexate in a murine leukemia model. Cancer Res., 36:4679-4686, 1976. 37. Kisliuk, R. L., Gaumont, Y., and Baugh, C. M. Polyglutamyl derivatives of 30. Cossins, E. A. Folates in biological materials. In: R. L. Blakley and S. J. folate as substrates and inhibitors of thymidylate synthetase. J. Biol. Chem., Benkovic (eds.), Folates and Pteridines, Vol. 1, pp. 2-60. New York: Wiley- 2*9:4100-4103,1974. Interscience, 1984. 38. Matthews, R. G., and Baugh, C. M. Interactions of pig liver methylenete- 31. McGuire, J. J., and Coward, J. K. Pteroylpolyglutamates: biosynthesis, trahydrofolate reducíasewith methylenetetrahydropteroylpolyglutamate sub degradation, and function. In: R. L. Blakely and S. J. Benkovic (eds.), Folates strates and with dihydropteroylpolyglutamate inhibitors. Biochemistry, 19: and Pteridines, Vol. 1, pp. 136-190. New York: Wiley-lnterscience, 1984. 2040-2045, 1980.

4209

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1989 American Association for Cancer Research. Relationship of Reduced Folate Changes to Inhibition of DNA Synthesis Induced by Methotrexate in L1210 Cells in Vivo

D. G. Priest, Marlene Bunni and F. M. Sirotnak

Cancer Res 1989;49:4204-4209.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/49/15/4204

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/49/15/4204. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1989 American Association for Cancer Research.